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Aversive responses of zebrafish to trypsin.a. Trypsin released by zebrafish in response to trypsin itself. Absorbance at 405 nm represents the yellow color generated by digestion of the s-2238 substrate by trypsin. (n = 6 independent experiments; For each experiment 1 fish was used; *p<0.001). b. A representative photograph showing the dispersion of larvae in the elongated mini-tanks after the larvae were introduced to the tank at the midpoint (arrowhead) followed by injection of test chemicals on the rightmost end of each tank. The test chemicals were BSA, RVV, collagen, trypsin, and L-cysteine. Note the movement of larvae to the left side of the tanks away from trypsin and L-cysteine. c. Quantification of dispersion as the % larvae displaced from the middle to the left side in the mini-tanks in response to BSA, RVV, Collagen, Trypsin, and L-Cysteine. (n = 6 experiments; *p<0.001; ns, non-significant). d.par2a transcripts were decreased markedly in the par2a knockdown zebrafish larvae (7 dpf). Results of RT-PCR with α-actin serving as the internal control and DNA size markers in lane M. Lanes 1–4 each represent mRNA from one animal. Control and par2a knockdown embryos were injected with sense cSO/cVMO and antisense aSO/cVMO hybrids for the piggyback knockdown. e. Intensity ratios for par2a and α-actin bands. par2a band intensity was divided by α-actin band intensity and plotted on Y-axis as a Relative ratio of intensities. Note the par2a knockdown ratios are significantly less than that of control samples (n = 6 individual larva; *p<0.001). f. Rescue of trypsin repelling in par2a knockdown larvae. Y-axis shows % larvae displaced from the middle to left side of the tank after trypsin treatment of control, par2a knockdown, and par2a rescued larvae (n = 6 experiments; *p<0.001; ns, non-significant).
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